This application relates to microgels for use in medical diagnosis, especially for the sequencing of nucleic acids, and to methods of making and using such gels.
DNA sequencing may be carried out using automated systems designed for laboratory application. Methods and apparatus for sequencing of DNA are described in U.S. Pat. Nos. 4,811,218; 4,823,007; 5,062,942; 5,091,652; 5,119,316 and 5,122,345, which are incorporated herein by reference.
The general methodology employed in these systems involves breaking up the sample DNA using restriction endonucleases; amplifying (for example with PCR) the restriction fragment of interest; combining the amplified DNA with a sequencing primer which may be the same as or different from the amplification primers; extending the sequencing primer in the presence of normal nucleotide (A, C, G, and T) and a chain-terminating nucleotide, such as a dideoxynucleotide, which prevents further extension of the primer once incorporated; and analyzing the product for the length of the extended fragments obtained. Analysis of fragments may be done by electrophoresis, for example on a polyacrylamide gel.
International Patent Publication No. WO93/00986 describes electrophoresis gels with a thickness of 25 to 250 microns. The gels are formed between two clamped-together plates, one of which is grooved to a depth equal to the desired gel thickness to form parallel tracks which are then filled with gel.
In performing a nucleic acid sequence analysis on a gel, the characteristics of the gel, including the size and thickness, impact the time and cost required to do the analysis. Since it is desirable to reduce the time and cost of sequencing analyses in order to improve the available of sequencing as a diagnostic tool, it would be advantageous to have a gel which permitted analysis of very small quantities of oligonucleotide fragments in a short period of time. It is an object of the present invention to provide such a gel.
It is a further object of the invention to provide single-use, disposable gel holders which are readily filled with gel to provide a gel for rapid analysis of small samples.
It is a further object of the present invention to provide a method of making gels which achieve high resolution of oligonucleotide fragments in a short period of time.
It is a further object of the invention to provide a method of evaluating a sample containing oligonucleotide fragments of various lengths.